After the reports by Kriaucionis et al. (Science 324:929-930 (2009)) and Tahiliani et al. (Science 324:930-935 (2009)) there has been a growing interest in detecting, locating and measuring hydroxymethylated nucleotides (hmNs), in particular, hydroxymethylated cytosines (hmCs), to better understand gene expression in eukaryotic cells, and in particular, mammalian cells. Unfortunately, sodium bisulfite sequencing does not differentiate between hmNs and methylated nucleotides (mNs). MspI which is an enzyme that is sometimes used along with HpaII to identify methylated cytosine (mC) also does not discriminate between hydroxymethylated and methylated DNA. Binding proteins used to immobilize fragments of DNA on an affinity substrate where the fragments contain a modified nucleotide do not differentiate between one or multiple modified nucleotides on the DNA fragment. This has meant that not only is it unknown where in the genome hmNs occur, but also how their presence and distribution varies in a genome according to the changing environment of the cell or the stage of a cell in differentiation.